Vitamin D Supplementation Ameliorates Metabolic Dysfunction in Patients with PCOS: A SystematicReview of RCTs and Insight into the Underlying Mechanism

Objective Evidence suggests that vitamin D deficiency correlated with metabolic disorders in women with polycystic ovary syndrome (PCOS). We conducted this systematic review and meta-analysis to evaluate the impact of vitamin D supplementation alone on glucose, lipid, and androgen parameters and inflammation biomarkers in women with PCOS. Methods Literature research was conducted in Pubmed, Embase, Web of Science, Clinical Trials, and Cochrane Library to identify relevant randomized controlled trials (RCTs) up to March 2020. The effect of vitamin D supplementation alone on women with PCOS was compared with administration of placebo. The systematic review and meta-analysis protocol was registered in the International Prospective Register of Systematic Reviews (Prospero) as number CRD42020157444. Results Thirteen randomized controlled trials with 824 patients in total were included. Serum FPG, fasting insulin, HOMA-IR, and VLDL-C were significantly decreased in the vitamin D group versus placebo. Vitamin D supplementation group also showed a significantly elevated level of QUICKI. No significant impact was seen on serum triglyceride, total-C, LDL-C, HDL-C, total testosterone, DHEAS, SHBG, or hs-CRP. Subgroup analysis demonstrated that oral vitamin D intake had significantly decreased serum triglyceride and total-C level in women with PCOS who have vitamin D deficiency (serum vitamin D < 20 ng/ml). Conclusion The findings of the present meta-analysis indicate that vitamin D supplementation exerted favorable effects among women with PCOS on glucose metabolism and lipid metabolism, especially in vitamin D deficient women, but had no significant effect on the androgenic profile or inflammation status.


Introduction
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting 6-10% of reproductive age women [1]. e main clinical manifestations of PCOS are irregular menstruation, polycystic ovarian morphology, hyperandrogenism, and infertility [2]. Women with PCOS are more prone to suffer from metabolic disorders including insulin resistance, dyslipidemia, and hypertension leading to increased long-term cardiovascular risk [3]. At present, treatments for patients with PCOS include life style interventions and drug therapy aiming at restoring menstruation and providing endometrial protection, decreasing androgen levels, and lowering insulin resistance [4]. Since the etiology and pathogenesis of the syndrome remain obscure, there is no specific therapy for this population. High prevalence of PCOS and its negative influence on both physical and psychological health of women have drawn important public health concerns.
Until now, metformin has been the first-line treatment for patients with PCOS who have insulin resistance, but associated side effects such as abdominal pain, diarrhea, or headache are common [5]. It is necessary to find a safe and economical treatment for these patients. Vitamin D is responsible for bone health by enhancing intestinal absorption of calcium [6]. Its importance in reproduction has become increasingly recognized over the past decade [7]. e receptor for vitamin D is expressed in several reproductive tissues including the ovary, uterus, and placenta [8,9]. Recent data from human studies suggests that vitamin D deficiency may be associated with reproductive disorders including PCOS. A meta-analysis comprising fourteen studies including a total of 2,262 women (1,150 patients with PCOS/1,162 controls) reported serum 25-hydroxyvitamin D was significantly lower in patients with PCOS than controls [8].
Vitamin D deficiency is defined as a 25-hydroxyvitamin (25-OH)D serum concentration of less than 20 ng/ml, while vitamin D insufficiency occurs at a level from 20 ng/ml to <30 ng/ml [10]. Numerous randomized controlled trials (RCT) have been conducted to evaluate the potential therapeutic effect of vitamin D supplementation in women with PCOS having vitamin D deficiency or insufficiency. Several published systematic review and meta-analyses have been performed, indicating that vitamin D oral supplementation had a little overall effect on metabolic status among patients with PCOS [11][12][13][14][15][16]. However, most of those articles included either non-RCTs or cosupplemented trials (with metformin or other nutrients), which may mask the real effect of vitamin D supplementation. Moreover, those published reviews tended to focus on one specific area, which may prevent us from getting a thorough understanding of the potential beneficial effects of vitamin D supplementation. Due to the conflicting results of vitamin D supplementation effects on glucose, lipid, androgenic, and inflammatory profile, we conducted a comprehensive systematic review and meta-analysis focusing on RCTs and studies only employing vitamin D supplementation alone without cosupplementation to reach a more convincing conclusion.

Registration.
e protocol for this systematic review was registered in the International Prospective Register of Systematic Reviews (Prospero) as number CRD42020157444 [17]. is systematic review and meta-analysis process and manuscript development complied with the PRISMA guidelines [18]. method was employed in case of data with identical measuring units; otherwise, standard mean difference (SMD) was adopted. Heterogeneity among studies was estimated by Cochran's Q test and I-squared. P < 0.10 with I 2 > 50% indicates statistical heterogeneity. When significant heterogeneity presented, the random-effects model of metaanalysis was applied. Publication bias was assessed by funnel plots.

Literature Search.
e online database search yielded 329 articles. After removing 147 duplicates, 182 articles were excluded by screening the title and abstract, and then, the remaining 61 articles were inspected carefully for eligibility. Studies without outcomes of interest (n � 3), study population (n � 3), and types of intervention (n � 32: concomitant metformin use, n � 5; concomitant OCP use, n � 1; concomitant other nutrients use, n � 26) not accordant with the inclusion criteria, studies without control group (n � 3), studies with incomplete data (n � 1), and studies without full text (n � 6) were excluded. Finally, 13 RCTs were selected according to the inclusion and exclusion criteria. e flowchart detailing selection of studies is presented in Figure 1.

Characteristics of Included RCTs.
ese papers were published from 2012 to 2019 and conducted in Iran [19][20][21][22][23][24][25][26], India [27], Austria [28], Venezuela [29], the United Kingdom [30], and the United States [31] A total of 824 women with PCOS aged 18-40 years were included, and the duration of intervention was 8 weeks, 12 weeks, or 24 weeks. e dose of vitamin D intake varied from 2000 IU/week to 50,000 IU/week. Essential features of trials included in the meta-analysis are summarized in Table 1. e overall risk of included trials was estimated to be low. e quality evaluation of included trials is shown in Figure 2.

Serum Vitamin D Levels.
Twelve studies reported significant increase in serum vitamin D level following supplementation. e baseline serum level of 25(OH)D in intervention group ranged from 3.5 ± 4.2 ng/ml to 19.55 ± 6.73 ng/ml, indicating that selected patients with PCOS were mostly vitamin D deficient. Combination of data extracted from thirteen studies revealed a significant increase in 25(OH)D concentrations in the vitamin D treatment group versus placebo (MD: 16.19 ng/ml, 95% CI: 13.30, 19.09) (Figure 3).

Publication Bias.
e funnel plots for serum vitamin D concentrations indicated a risk for lack of reporting on negative effect of vitamin D supplementation. e funnel plots for the rest of the indices showed there was no significant publication bias. However, for each result, the number of studies of meta- Full-text articles screened N = 61 Included RCTs: 13

Discussion
Several meta-analyses have been conducted concerning the effect of vitamin D supplementation on metabolic biomarkers of women with PCOS, suggesting variable beneficial effects, but the results remained conflicting. Moreover, the data of non-RCTs and cosupplementation trials were combined in most of these reviews. In view of the increasing number of RCTs regarding this topic, we conducted the present meta-analysis of RCTs focusing on the effect of vitamin D intake alone without cosupplementation compared with placebo to reach more convincing conclusions. e results of our meta-analysis revealed that vitamin D oral intake alone improved insulin resistance parameters and reduced inflammation in patients with PCOS. Furthermore, subgroup analysis showed that lipid metabolism was also improved in vitamin D deficient group. No effects were found on serum androgen levels or inflammation status.
Vitamin D deficiency is very prevalent in women with PCOS. A recent cross-sectional study demonstrated that, compared with fertile controls, significantly lower vitamin D levels were present in women with PCOS (mean 25(OH)D of 64.5 nmol/l vs. 49.0 nmol/l, resp.); meanwhile, higher HOMA-   IR and lipid abnormalities are associated with deficient vitamin D levels [32]. e recommended dose of vitamin D supplementation by the Endocrine Society Guidelines is 50,000 IU once weekly for eight weeks [33]. In our meta-analysis, most of the included patients with PCOS had insufficient vitamin D level (<30 ng/ml). After vitamin D supplementation, serum 25(OH)D concentrations in patients with PCOS increased significantly with a mean difference of 16.19 ng/ml (95% CI: 13.30, 19.09). e heterogeneity of this response is high (I 2 � 95%, P < 0.00).
Evidence from our meta-analysis showed that vitamin D supplementation resulted in lowering blood fasting glucose levels in addition to improving insulin resistance, as seen by a significant decrease in serum fasting insulin and HOMA-IR along with a slight increase in QUICKI. e results of our metaanalysis are in contrast with earlier ones by other researchers. Xue et al. [11] and Fang et al. [12] failed to find a positive effect of vitamin D supplementation on glucose metabolism in women with PCOS. Additionally, in a more recent metaanalysis, vitamin D supplementation was found to affect glucose and HOMA-IR significantly only when cosupplemented with other nutrients [16]. Several reasons may be underlying these differences. First, the meta-analysis by Lagowska et al. incorporated RCT studies that included diverse means of intervention with different kinds of micronutrients in addition to vitamin D, which may explain more variable treatment effects [16]. Second, prior meta-analyses did not exclude studies in which commonly used medications to treat PCOS such as metformin and OCPs were used [12,13,16]. e use of such medications known to ameliorate insulin resistance in the placebo control group may diminish the observed effects of vitamin D intervention. ird, prior meta-analyses have also included a number of single-arm before-after studies [11], which could make the pooled results less robust. To further study the optimum way of oral vitamin D administration, we conducted subgroup analysis revealing that daily intake could                             reduce serum glucose and insulin level, while low-dose intake could alleviate insulin resistance conditions. e potential mechanism of the effect of vitamin D on glucose metabolism may be the result of both genomic and nongenomic effects. Increased25(OH)D enhances VDR signaling helping pancreatic β-cells restore function through BRD7/PBAF pathway [34]. e 1, 25(OH)2D-VDR complex binds to the vitamin D response element of the insulin receptor in tissue improving insulin responsiveness for glucose transport and suppressing the release of proinflammatory cytokines, which are believed to mediate IR [34]. Moreover, its influence on the extracellular and intracellular calcium regulation is vital for the modulation of glucose transport in target tissues [35].
Notably, decline in serum triglycerides, total-C, and VLDL-C concentration were significant among patients with PCOS who are deficient in vitamin D in our meta-analysis. is finding is in accordance with the significant improvement of serum triglycerides level observed in the meta-analysis by Xue et al. [11]. However, in Xue's article, this effect was observed with low dose vitamin D supplementation (<50,000 IU/week) rather than high dose. However, in our study, subgroup analysis showed that a low dose of vitamin D supplementation had no impact on triglycerides or total-C level, which means that highdose vitamin D intake may be more beneficial for lipid metabolism. A clarified mechanism that can explain the beneficial effects of vitamin D on lipid metabolism is that increased intracellular calcium in the liver leads to the stimulation of microsomal triglyceride transfer protein, which participates in the formation and secretion of VLDL, resulting in decreased circulating levels of serum total-C [25]. Additionally, in a randomized controlled trial, it was shown that serum VEGF levels and TGF-β1 to soluble endoglin (sENG) ratio is significantly decreased by vitamin D supplementation in women with PCOS [31,36]. e decrease in VEGF and TGF-β1 : sENG ratio correlated with the decrease in triglycerides in the PCOS group. Increased circulating TGF-β1 levels have been associated with various cardiometabolic conditions including obesity [37], diabetes [38], and coronary artery disease [39]. It has been suggested that higher levels of insulin, LH, and androgens increase the concentration of VEGF, leading to abnormally increased vascularity in the ovary, which may exacerbate anovulation and subfertility. TGF-β1 upregulates the secretion of VEGF, and the resulting angiogenic imbalance may play a role in the development of metabolic syndrome and cardiovascular disease in women with PCOS [40].
In this meta-analysis, we did not find any improvement in serum total testosterone, DHEAS, or SHBG level. Similar findings were reported in the meta-analysis by Azadi-Yazdi et al. [14]. In their study, significant decrease in serum testosterone concentration was only found in single-arm before-after studies, but not in high-quality RCTs. Meanwhile, in the current meta-analysis, we found that serum hs-CRP level did not change significantly after vitamin D supplementation. Previously, a meta-analysis conducted by Akbari et al. [15] reported that vitamin D supplementation resulted in an improvement in hs-CRP of women with PCOS, but in this article, the significant decrease was evident after including three trials examining the anti-inflammatory effect of vitamin D and other nutrition. Our meta-analysis demonstrated that vitamin D supplementation alone did not influence hs-CRP level among women with PCOS.
Our meta-analysis has several strengths. It includes comprehensive research of RCTs and strict inclusion of high-quality studies, in which vitamin D intervention was given alone.
is allowed for a more focused analysis of vitamin D effects in PCOS. Our work also has some  limitations.
ere is considerable heterogeneity between studies with variable race/ethnicity and age. In addition, baseline vitamin D status, vitamin D dose, and formulation and duration of treatment of vitamin D vary between studies and are an additional source of confounding. Moreover, most of the trials included were conducted in Iran, where women are covered by clothes resulting in less exposure to sunlight, so whether the benefits of vitamin D supplementation can be generalizable to women with PCOS from all over the world is still an open question. In addition, considering the limited number of high-quality RCTs and small sample size, the conclusions of the present metaanalysis should be extrapolated with caution. However, as an inexpensive and safe treatment option, vitamin D supplementation can be implemented in women with PCOS who   16 International Journal of Endocrinology have vitamin D deficiency. Our study may shed light on the potential mechanisms behind those discovered benefits that vitamin D deficiency may be a codeterminant factor in metabolism disorder. Taken together, vitamin D supplementation appears to influence several aspects of clinical features present in women with PCOS ( Figure 9).

Conclusion
Our systematic review showed that oral intake of vitamin D supplementation attenuated insulin resistance and hyperlipemia, but not androgenic profile or inflammatory markers in women with PCOS who have vitamin D deficiency. Several potential mechanisms may be underlying these beneficial effects of vitamin D on clinical features of PCOS, and further research is needed to explore the complex role of vitamin D in different pathways of this metabolic disorder.

Data Availability
e data used in the study are available upon request to the corresponding author.

Conflicts of Interest
e authors report no conflicts of interest related to this work.